Actin is an essential protein in all eukaryotic cells. When polymerized into filaments, actin plays an important role in cell motility and in maintaining cell shape. Not surprisingly, a wide variety of diseases lead to a breakdown in actin filaments. Regardless of the etiology of the disease, breakdown of actin filaments can have life threatening consequences, such as in the case of metastatic cancer and muscular dystrophy. Thus, understanding the regulation of actin filaments is essential to our understanding of the molecular mechanisms underlying many diseases. This information will hopefully improve patient care and survivability. The long term goal of this project is to understand how actin monomer incorporates into existing actin filaments and whether this process differs between isoforms of actin. An in vitro system will be used to study the incorporation of actin into myofibrils. Actin will be translated in a reticulocyte lysate in the presence of 35S Met or purified from muscle, labeled with biotin or a fluorescent probe and then added to the reticulocyte lysate. Myofibrils will be added and incubated to allow actin to incorporate. Myofibrils will then be collected by centrifugation and incorporated proteins analyzed by gel electrophoresis or microscopy. The following questions will be addressed: 1. How does the myofibrillar architecture influence the incorporation of actin into the myofibril? 2. Do isoforms of actin show a similar or different pattern of incorporation? 3. Can muscle and non-muscle actin isoforms sort themselves between sarcomeric and non-sarcomeric filaments? 4. What role does the protein binding domains of actin have in the incorporation process?